The importance of the PI3Kinase-Akt-Tsd/Tsc2-mTOR signaling pathway in the regulation of both cell size and cell growth has become apparent in recent years. The seminal observations that initiated this explosive increase in our understanding were made in Drosophila 4 years ago. Although much progress has been made in mammalian systems on this pathway as well, Drosophila continues to offer major advantages and insights due to the ease of genetic manipulation, and relatively low costs. In addition, recently the technological approach of whole genome RNAi screens in Drosophila has provided the opportunity for pathway analysis that cannot be approached in mammalian systems. This approach is robust and reliable, and is the primary tool we will use to explore critical questions on Tsc1/Tsc2-Tor-S6K signaling in four specific aims. In Aim 1 and Aim 2, two small scale - hypothesis driven - RNAi screen will be performed to identify a crucial missing links in this pathway: The GEF(s) for Rheb and kinases/phosphatases affecting AMPK_ phosphorylation and activity. In Aim 3, we will identify core components involved in Akt phosphorylation and its regulation by a feedback mechanism. We will distinguish those that affect both S6K and Akt,and those that affect Akt only. In Aim 4, a comprehensive genome-wide RNAi screen will be performed to identify regulators acting downstream of Tsc1/Tsc2 on S6K phosphorylation. Findings will be explored and confirmed through biochemical and genetic analyses in vivo in the fly,and also translated to mammalian systems in concert with the other projects of this PPG. The improved understanding of the wiring of this signaling pathway will provide both fundamental insight and the opportunity for therapeutic intervention.